A ternary complex of hydroxycinnamoyl-CoA hydratase-lyase (HCHL) with acetyl-CoA and vanillin gives insights into substrate specificity and mechanism

TY - JOUR

T1 - A ternary complex of hydroxycinnamoyl-CoA hydratase-lyase (HCHL) with acetyl-CoA and vanillin gives insights into substrate specificity and mechanism

AU - Bennett, Joseph P.

AU - Bertin, Lucille

AU - Moulton, Benjamin

AU - Fairlamb, Ian J. S.

AU - Brzozowski, A. Marek

AU - Walton, Nicholas J.

AU - Grogan, Gideon

PY - 2008/9/1

Y1 - 2008/9/1

N2 - HCHL (hydroxycinnamoyl-CoA hydratase-lyase) catalyses the biotransformation of feruloyl-CoA to acetyl-CoA and the important flavour-fragrance compound vanillin (4-hydroxy-3-methoxy-benzaldehyde) and is exploited in whole-cell systems for the bio-conversion of ferulic acid into natural equivalent vanillin. The reaction catalysed by HCHL has been thought to proceed by a two-step process involving first the hydration of the double bond of feruloyl-CoA and then the cleavage of the resultant beta-hydroxy thioester by retro-aldol reaction to yield the products. Kinetic analysis of active-site residues identified using the crystal structure of HCHL revealed that while Glu-143 was essential for activity, Ser-123 played no major role in catalysis. However, mutation of Tyr-239 to Phe greatly increased the K-M for the substrate binding. Structures of WT (wild-type) HCHL and of the S123A mutant, each of which had been co-crystallized with feruloyl-CoA, reveal a subtle helix movement upon ligand binding, the consequence of which is to bring the phenolic hydroxyl of Tyr-239 into close proximity to Tyr-75 from a neighbouring subunit in order to bind the phenolic hydroxyl of the product vanillin, for which electron density was observed. The active-site residues of ligand-bound HCHL display a remarkable three-dimensional overlap with those of a structurally unrelated enzyme, vanillyl alcohol oxidase, that also recognizes p-hydroxylated aromatic substrates related to vanillin. The data both explain the observed substrate specificity of HCHL for p-hydroxylated cinnamate derivatives and illustrate a remarkable convergence of the molecular determinants of ligand recognition between the two otherwise unrelated enzymes.

AB - HCHL (hydroxycinnamoyl-CoA hydratase-lyase) catalyses the biotransformation of feruloyl-CoA to acetyl-CoA and the important flavour-fragrance compound vanillin (4-hydroxy-3-methoxy-benzaldehyde) and is exploited in whole-cell systems for the bio-conversion of ferulic acid into natural equivalent vanillin. The reaction catalysed by HCHL has been thought to proceed by a two-step process involving first the hydration of the double bond of feruloyl-CoA and then the cleavage of the resultant beta-hydroxy thioester by retro-aldol reaction to yield the products. Kinetic analysis of active-site residues identified using the crystal structure of HCHL revealed that while Glu-143 was essential for activity, Ser-123 played no major role in catalysis. However, mutation of Tyr-239 to Phe greatly increased the K-M for the substrate binding. Structures of WT (wild-type) HCHL and of the S123A mutant, each of which had been co-crystallized with feruloyl-CoA, reveal a subtle helix movement upon ligand binding, the consequence of which is to bring the phenolic hydroxyl of Tyr-239 into close proximity to Tyr-75 from a neighbouring subunit in order to bind the phenolic hydroxyl of the product vanillin, for which electron density was observed. The active-site residues of ligand-bound HCHL display a remarkable three-dimensional overlap with those of a structurally unrelated enzyme, vanillyl alcohol oxidase, that also recognizes p-hydroxylated aromatic substrates related to vanillin. The data both explain the observed substrate specificity of HCHL for p-hydroxylated cinnamate derivatives and illustrate a remarkable convergence of the molecular determinants of ligand recognition between the two otherwise unrelated enzymes.

KW - aldolase

KW - crotonase

KW - enoyl-CoA hydratase (ECH)

KW - hydroxycinnamoyl-CoA hydratase-lyase (HCHL)

KW - ligand complex

KW - vanillin

KW - 1.8 ANGSTROM RESOLUTION

KW - FERULIC ACID

KW - CROTONASE SUPERFAMILY

KW - ALCOHOL OXIDASE

KW - COENZYME-A

KW - PSEUDOMONAS-FLUORESCENS

KW - NATURAL-PRODUCT

KW - ENZYME

KW - BIOSYNTHESIS

KW - METABOLISM

UR - http://www.scopus.com/inward/record.url?scp=50949084595&partnerID=8YFLogxK

U2 - 10.1042/BJ20080714

DO - 10.1042/BJ20080714

M3 - Article

VL - 414

SP - 281

EP - 289

JO - Biochemical journal

JF - Biochemical journal

SN - 0264-6021

IS - 2

ER -